Antibodies are highly useful reagents in biotechnological and biomedical applications, yet recombinant antibodies have their limitations. These include the complex architecture of their antigen- binding sites, which complicates the generation of synthetic antibody libraries, and major patent restrictions on recombinant antibody development. As a result, there has been an intensive search for alternatives to antibodies in the form of non-immunoglobulin (Ig) scaffolds such as ankyrin repeats, fibronectins, lipocalins and protein A. However, only a few of these artificial scaffolds have actually yielded specificities towards different types of targets, possibly because none of them are natural antigen receptors. Indeed, the only known natural non-Ig antigen receptors are the variable lymphocyte receptors (VLRs) of lamprey and hagfish. VLRs consist of highly diverse leucine-rich repeat modules, which are assembled by DNA recombination via gene conversion, resulting in a potential repertoire of over 1014 unique receptors. Lamprey immunized with various antigens respond by production of high avidity plasma VLRs specific for the antigen. VLRs are thus ideal candidates to serve as non-Ig single chain antibodies in biotechnology. We have developed a high-throughput platform for the selection of recombinant antigen-binding VLRs from large VLR libraries (>107 independent clones) using yeast surface display technology and a customized yeast display vector. We have isolated VLRs with low micromolar affinity to hen egg lysozyme (HEL) and low nanomolar affinity to R-phycoerythrin. One round of in vitro affinity maturation decreased by 13-fold the dissociation constant of an HEL-binding VLR, and a VLR-HEL complex has been crystallized. We propose to establish the biotechnological potential of monoclonal VLRs as a new class of molecular recognition molecules. Our goals are: 1. Clone VLRs that specifically bind diverse ligands representing the antigenic world, including proteins, carbohydrates and whole bacteria. Both naove and immune libraries will be constructed and screened for binders by yeast surface display. 2. Characterize the biochemical and structural properties of VLR-interactions using surface plasmon resonance combined with X-ray crystallography. 3. Engineer high affinity ligand-binding VLRs by random or targeted mutagenesis. Collectively, these studies will define the affinity range of VLRs for diverse antigens, determine whether the naove VLR repertoire is sufficiently diverse to allow isolation of specific binders from nonimmune libraries, define the structural basis for antigen recognition by VLRs, and establish efficient strategies for affinity maturation of these novel antigen receptors. PUBLIC HEALTH RELEVANCE: Although mammalian antibodies are highly useful reagents in biotechnology, they have certain limitations related to their size and complexity. We propose to develop high affinity recombinant antibodies from the sea lamprey as a completely new class of recognition molecules for diagnostic, analytic, and biosensor applications in biotechnology.